Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/475—Growth factors; Growth regulators
  • C07K14/485—Epidermal growth factor [EGF] (urogastrone)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• peptide growth factors influence epithelial and epidermal cells through autocrine and paracrine mechanisms. They also play important roles in normal wound healing in tissues such as skin, cornea and gastrointestinal tract and all share substantial amino acid sequence homology including the conserved placement of three intra-chain disulfide bonds. In addition, all the factors of this family bind to a 170,000 molecular weight transmembrane glycoprotein receptor and activate the tyrosine kinase activity in the receptor's cytoplasmic domain (Buhrow, S.A. et al., J. Bio.Chem.. 258:7824-7826 (1983)).
• novel mature polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
• the polypeptides of the present invention are of human origin.
• processes for producing such polypeptide by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention.
• a polynucleotide encoding a polypeptide of the present invention may be obtained from human brain and early stage brain tissue.
• the polynucleotide of this invention was discovered in a human osteoclastoma cDNA library. It has homology to the characteristic EGF domains. It contains an open reading frame encoding a polypeptide of 443 amino acids with a putative leader sequence of 24 amino acids.
• HCABA58X exhibits the highest degree of homology at the amino acid level to human extracellular protein with 51% identity and 30% similarity over a 387 amino acid stretch.
• Northern blot analysis of this protein shows high levels of expression in heart and kidney tissue with the transcript being approximately 2 kb.
• polynucleotide encoding a polypeptide encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
• the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 (SEQ ID NO:2) as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 (SEQ ID NO:2) .
• Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
• the polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.
• polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
• polypeptides of the present invention include the polypeptide of SEQ ID NO:2 (in particular the mature polypeptide) as well as polypeptides which have at least 70% similarity (preferably at least 70% identity) to the polypeptide of SEQ ID NO:2 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of SEQ ID NO:2 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of SEQ ID NO:2 and also include portions of such polypeptides with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
• similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
• Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full- length polypeptide by peptide synthesis,- therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
• the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
• promoter for example, LTR or SV40 promoter, the E. coli. lac or trp. the phage lambda P L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
• the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
• the vector may also include appropriate sequences for amplifying expression.
• constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
• the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
• Microbial cell ⁇ employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those skilled in the art.
• the EGF receptor gene represents the cellular homolog of the v-erb-B oncogene of avian erythroblastosis virus.
• Over expres ⁇ ion of the EGF-receptor or deletion of kina ⁇ e regulatory segments of the protein can bring about tumorigenic transformation of cells (Manjusri, D. et al., Human Cytokines. 364 and 381 (1991) ) .
• conditions suitable for treatment with the polypeptide of the present invention include chronic conditions, such as chronic ulcers, diabetic ulcers, other non-healing (trophic) conditions, to treat Marfan syndrome, to promote hair follicular development, to stimulate growth and differentiation of various epidermal and epithelial cells in vivo and in vitro and to stimulate embryogenesis.
• chronic conditions such as chronic ulcers, diabetic ulcers, other non-healing (trophic) conditions
• Marfan syndrome to promote hair follicular development
• various epidermal and epithelial cells in vivo and in vitro and to stimulate embryogenesis.
• Certain disease pathologies may be partially or completely ameliorated by the systemic clinical administration of the HCABA58X growth factor.
• This administration can be in the form of gene therapy (see below) ,- or through the administration of peptides or proteins synthesized from recombinant constructs of HCABA58X DNA or from peptide chemical synthesis (Woo, et al. , Protein Engineering 3:29-37 (1989) .
• This invention provides a method for identification of HCABA58X receptors.
• the gene encoding a receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al. , Current Protocols in Immun., 1(2), Chapter 5, (1991)) .
• expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to HCABA58X, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to HCABA58X.
• Transfected cells which are grown on glas ⁇ ⁇ lides are exposed to labeled HCABA58X, which can be labeled by a variety of means including iodination or inclusion of a recognition site for a site- ⁇ pecific protein kina ⁇ e. Following fixation and incubation, the slides are subjected to auto-radiographic analysis. Positive pools are identified and sub-pools are prepared and re-transfected using an iterative sub-pooling and re-screening process, eventually yielding a single clone that encodes the putative receptor.
• labeled ligand can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule.
• Cros ⁇ -linked material is resolved by PAGE and exposed to X-ray film.
• the labeled complex containing the ligand-receptor can be excised, resolved into peptide fragments, and subjected to protein microsequencing.
• the amino acid sequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.
• Another assay for identifying potential antagoni ⁇ t ⁇ ⁇ pecific to the receptor ⁇ to the polypeptide of the present invention is a competition assay which comprises isolating plasma membranes which over-express a receptor to the polypeptide of the present invention, for example, human A431 carcinoma cells.
• Serially diluted test sample in a medium (volume is approximately 10 microliters) containing 10 nM l25 I-HCABA58X is added to five micrograms of the plasma membrane in the presence of the potential antagonist compound and incubated for 4 hours at 4°C.
• the reaction mixtures are diluted and immediately passed through a millipore filter.
• the filters are then rapidly washed and the bound radioactivity is measured in a gamma counter.
• the amount of bound HCABA58X is then measured.
• a control assay is also performed in the absence of the compound to determine if the antagonist ⁇ reduce the amount of bound HCABA58X.
• Potential antagonist compounds include an antibody, or in some cases, an oligopeptide, which binds to the polypeptide.
• a potential antagonist may be a closely related protein which binds to the receptor which is an inactive forms of the polypeptide and thereby prevent the action of the polypeptide of the present invention.
• the antagonists may be employed to treat neoplasia, for example, cancers and tumors. It is known that inhibition of secretion or production of members of the EGF family by tumor cells in mice causes regression of tumors, since these protein ⁇ ⁇ timulate induction of DNA ⁇ ynthesis in all cells including neoplastic cells.
• the reporter antibody linked to horseradish peroxidase is now placed in the di ⁇ h resulting in binding of the reporter antibody to any monoclonal antibody bound to polypeptides of the present invention. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of protein present in a given volume of patient sample when compared against a standard curve.
• PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome.
• sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner.
• Other mapping strategies that can similarly be used to map to its chromosome include in situ hybridization, prescreening with labeled flow-sorted chromosome ⁇ and preselection by hybridization to construct chromosome specific-cDNA libraries.
• Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromo ⁇ omal location in one ⁇ tep.
• Thi ⁇ technique can be used with cDNA as short as 50 or 60 bases. For a review of this technique, see Verma et al., Human Chromosomes: a Manual of Basic Technique ⁇ , Pergamon Press, New York (1988) .
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This assumes 1 megabase mapping resolution and one gene per 20 kb) .
• polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
• These antibodies can be, for example, polyclonal or monoclonal antibodies.
• the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expres ⁇ ion library. Variou ⁇ procedure ⁇ known in the art may be used for the production of such antibodies and fragments.
• Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodies can then be used to isolate the polypeptide from tis ⁇ ue expre ⁇ sing that polypeptide.
• Ligase refers ⁇ to the process of forming phosphodiester bonds between two double ⁇ tranded nucleic acid fragments (Maniatis, T. , et al., Id., p. 146) . Unles ⁇ otherwi ⁇ e provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
• ligase T4 DNA ligase
• the DNA sequence encoding HCABA58X is initially amplified using PCR oligonucleotide primers corresponding to the 5' sequences of the processed protein and the sequence ⁇ 3' of the HCABA58X gene.
• the 5' oligonucleotide primer ha ⁇ the sequence 5' GATCGCATGCTCCCCTGCGCCTCCTG 3' (SEQ ID NO:3) contains a SphI restriction enzyme site followed by 17 nucleotides of HCABA58X coding sequence.
• the 3' sequence 5' GACTGGATCCGAAGGTGTAGGCCCCTAC 3' contains complementary sequences to a BamHI site and i ⁇ followed by 18 nucleotides of HCABA58X.
• the DNA sequence encoding the HCABA58X protein, ATCC # 97376, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the gene.
• the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene.
• the polyhedrin sequences are flanked at both side ⁇ by viral sequences for the cell-mediated homologous recombination of co- transfected wild-type viral DNA.
• Many other baculovirus vectors could be used ⁇ uch a ⁇ pAc373, pRGl, pVL94l and pAcIMl (Luckow, V.A. and Summer ⁇ , M.D., Virology, 170:31-39) .
• HCABA58X For expression of the recombinant HCABA58X, COS cells are transfected with the expression vector by DEAE-DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Press, (1989)) .
• the expression of the HCABA58X HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Pres ⁇ , (1988)) .
• Cell ⁇ are labelled for 8 hours with 35 S-cysteine two days post transfection.
• Fibroblasts are obtained from a subject by skin biopsy.
• the resulting tissue is placed in ti ⁇ ue-culture medium and ⁇ eparated into small pieces. Small chunks of the tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask.
• the fla ⁇ k i ⁇ turned up ⁇ ide down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom of the flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin, is added. This i ⁇ then incubated at 37°C for approximately one week.
• fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin

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• 1996
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